The device of the invention addresses miniaturization of reaction chambers for the purpose of using smaller quantities of sample and reagents. Smaller chambers often require a working surface area and means to prevent evaporation of the small volume of reagents, either due to elevated temperatures or prolonged reaction periods. Covering a working surface area by stretching out a small liquid volume results in a generally planar chamber, as opposed to commonly used U-shaped, or V-shaped cuvettes. The ratio of working surface area to volume is significantly increased when the reaction area is stretched out, but so is the surface area available for evaporation at the interface between the aqueous-based reagent liquids and air. Covers and seals over the liquid flow path of complex reactions may be more critical than those used in immunodiagnostic test strips, where evaporation does not affect test results because reaction times are usually quite rapid.
Reaction devices are described in U.S. Pat. Nos. 5,346,672; 5,188,963 and 5,451,500, which patents are herein incorporated by reference. One device controls evaporation by sealing a cover to a microscope slide. The others are method and apparatus inventions that integrate the sample preparation and detection of nucleic acids in biological specimens by flowing a series of reagents and washes over a thin matrix or through a thin chamber.
It is well known in the field of molecular biology that the more sensitive is the method of molecular detection, the fewer number of copies of the molecular target are needed for detection. The need to amplify either the target sequence or the signal arises when the specimen material does not contain enough molecules to detect above a complex genetic background or when analyzing the fine sequence detail of a nucleic acid sequence. U.S. Pat. No. 5,382,511, herein incorporated by reference, uses enzymes such as polymerase or ligase, separately or in combination, to repeatedly generate more copies of a target nucleic acid sequence from immobilized samples by primer extensions to incorporate new nucleotides or by ligations of adjacent complementary oligonucleotides, wherein each template generates more copies and the copies may themselves become template. By melting complementary strands of nucleic acids, the original strand and each new strand synthesized are potential templates for repeated primer annealing or ligation reactions to make and expand the number of specific, amplified products. More thermostable ligases and polymerases with reverse transcriptase activity are commercially available and increase the choice of enzymes and combination of reactions for immobilized sample amplification applications. Immobilized Sample Amplification, ISA, can either be primer extensions in one direction for linear amplification, or in opposing directions, for geometric amplification, and the like, when replicas are generated from a target within the immobilized biological specimen.
Although the applicant first achieved Immobilized Sample Amplification in a chamber made by sealing two glass cover slips together with rubber cement; the technique using rubber cement or nail polish is messy, labor-intensive and prone to failure during thermocycling. The Gene Cone chamber (U.S. Pat. No. 5,346,672, GeneTec Corporation, Durham, N.C.) was invented by the applicant and injection-molded as a plastic part that is adhered to a glass slide to form a reaction chamber; the plastic part has an opening for adding reagents to the chamber and then the opening is closed. Another thin chamber, the Amplicover is made by pressing a flat molded part against a glass slide by a spring clamping mechanism after the reagent is added to the specimen on the slide (In Situ System 1000, Perkin-Elmer, Norwalk, Conn.). Reagents spill out at the edges of the Amplicover before a gasket-like seal is formed by the clamp. The Gene Cone chamber, unlike the Amplicover, has a port to allow for removal and addition of subsequent reagents and wash treatments. However, it is difficult to dispense into a Gene Cone chamber without touching its surface and it is not practical to remove liquid from its thin spaces.
One way to miniaturize reactions is to make wells smaller and closer together; however it becomes more difficult to insure that only the reagent or sample intended for each well is delivered to it. Another way to miniaturize reactions is to increase the number of tests per reaction volume by localizing different tests in an array with a capability to discriminate individual signals from each component of the array. Thus, an positional array with 100 components generates 100 signals in the same volume that previously generated a single signal and effectively reduces the volume of reagent required per test 100-fold.
Attempts to miniaturize reaction chambers are confronted with the problem of how to introduce liquids into, or remove them from, reaction chambers that comprise thin spaces and, in particular, how to keep the working area liquid-covered and bubble-free. Among the factors causing bubbles to arise in liquids are irregular flow patterns, temperature and pressure changes and gaseous discharge at electrodes. It is well known that evacuating liquids and removing bubbles from a thin space is difficult. Filling and moving fluids in spaces thin enough to exhibit capillarity often are not predictable by standard engineering fluid dynamics.